miR-34a is associated with clinical outcome of colorectal cancer patients

Aberrant expression of microRNA-34a (miR-34a) has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in colorectal cancer (CRC) has not been completely clarified. In the current study, we have investigated the clinical significance of miR-34a. MiR-34a expression in forty-three cases of colorectal cancer tissues decreased significantly compared to that in the adjacent non-tumorous colorectal tissues (P＜0.05), as detected by real-time quantitative RT-PCR (qRT-PCR). Significantly, the expression of miR-34a was correlated with infiltration depth and clinical TNM stage (P <0.05). The miR-34a however had no correlation with other features, such as age, gender, site, tumor sizes, lymph node metastasis, serous membrane infiltration ( all P> 0.05). MiR-34a is a tumor suppressor miRNA that plays a vital role in the oncogenesis and progression of colorectal cancer. This study suggests that miR-34a may be a new tumor marker or prognostic factor in colorectal cancer. The strategies to increase miR-34a level might be a critical targeted therapy for CRC in the future.     

Discovery of core gene families associated with liver metastasis in colorectal cancer and regulatory roles in tumor cell immune infiltration

In this study, we aimed to uncover genes that drive the pathogenesis of liver metastasis in colorectal cancer (CRC), and identify effective genes that could serve as potential therapeutic targets for treating with colorectal liver metastasis patients based on two GEO datasets. Several bioinformatics approaches were implemented. First, differential expression analysis screened out key differentially expressed genes (DEGs) across the two GEO datasets. Based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, we identified the enrichment functions and pathways of the DEGs that were associated with liver metastasis in CRC. Second, immune infiltration analysis identified key immune signature gene sets associated with CRC liver metastasis, among which two key immune gene families (CD and CCL) identified as key DEGs were filtered by protein-protein interaction (PPI) network. Some of the members in these gene families were associated with disease free survival (DFS) or overall survival (OS) in two subtypes of CRC, namely COAD and READ. Finally, functional enrichment analysis of the two gene families and their neighboring genes revealed that they were closely associated with cytokine, leukocyte proliferation and chemotaxis. These results are valuable in comprehending the pathogenesis of liver metastasis in CRC, and are of seminal importance in understanding the role of immune tumor infiltration in CRC. Our study also identified potentially effective therapeutic targets for liver metastasis in CRC including CCL20, CCL24 and CD70.     

Loss of E-cadherin promotes the growth,invasion and drug resistance of colorectal cancer cells and is associated with liver metastasis

The recent studies indicated that the epithelial cell adhesion molecule E-cadherin is a well-recognized molecule that is important in cell adhesion. To further investigate the molecular basis of this notion, we used small-interfering RNA to inhibit E-cadherin function and found that loss of E-cadherin promoted Colorectal cancer cell growth, invasion and drug resistance through induction of β-catenin nuclear translocation and epithelial-to-mesenchymal transition. Further analysis of E-cadherin expression with clinicopathologic parameters showed that E-cadherin expression decreased in Colorectal cancer patients who developed liver metastasis (P = 0.043). These findings indicate that E-cadherin loss in tumors contributes to progression and metastatic dissemination. Thus, E-cadherin can act as a central modulator of the cell biological phenotypes and a potential prognostic marker in Colorectal cancer.     

Serum level of soluble 70-kD heat shock protein is associated with high mortality in patients with colorectal cancer without distant metastasis

Many findings indicate that measuring the serum concentration of soluble 70-kD heat shock protein (soluble HSP70) may provide important information in cardiovascular, inflammatory, and pregnancy-related diseases; however, only scarce data are available in cancer. Therefore, using a commercial ELISA kit, we measured soluble HSP70 concentration in the sera of 179 patients with colorectal cancer. We investigated the relationship between soluble HSP70 concentration and mortality, during 33.0 (24.4–44.0) months long follow-up. High (>1.65 pg/ml, median concentration) soluble HSP70 level was a significant (hazard ratio: 1.88 (1.20–2.96, p = 0.005) predictor of mortality during the follow-up period. When we compared the soluble HSP70 levels in patients with non-resected primary tumors as compared to those who were recruited into the study 4–6 weeks after the tumor resection they were found to be significantly (p = 0.020) higher in the former group. Since the patients with non-resected primary tumors had also distant metastasis and died early, we limited the further analysis to 142 patients with no distant metastasis at the beginning of the follow-up. This association remained significant even after multiple Cox-regression analysis had been performed to adjust the data for age and sex (p = 0.028); age, sex, and TNM-T stage (p = 0.041); age, sex, and TNM-N stage (p = 0.021); age, sex, and histological grade (p = 0.023); or age, sex, and tumor localization (p = 0.029). Further analysis showed that the significant association between high HSP70 levels and poor survival is in the strongest in the group of <70-year-old female patients (HR: 5.52 (2.02-15.15), p = 0.001), as well as in those who were in a less advanced stage of the disease at baseline. These novel findings indicate that the serum level of soluble HSP70 might prove a useful, stage-independent prognostic marker in colorectal cancer without distant metastasis.     

Proteomic profiling of proteins associated with lymph node metastasis in colorectal cancer

Lymph node metastasis (LNM) is associated with poor prognosis in colorectal cancer (CRC). The presence or absence of lymph node metastases is a strong independent prognostic factor for CRC survival. Investigation of proteins associated with the process of lymph node metastasis (LNM) is crucial for understanding of the molecular mechanisms underlying the LNM process and for predicting the CRC prognosis. In the present study, proteins from CRC tissues and adjacent normal mucosa (NMC) were examined using two‐dimensional gel electrophoresis coupled with MALDI‐TOF‐MS. The expression levels of Ferritin Heavy Chain (FHC) were decreased in LNM CRC as compared to those in non‐LNM CRC, while the expression of Cathepsin D and Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1) were increased in LNM CRC. The results were confirmed by Western blotting and immunohistochemical staining. Furthermore, in vitro cell invasion assay showed that the overexpression of UCH‐L1 through gene transfection increased the invasive ability of HCT8 cells, suggesting that UCH‐L1 is not only a biomarker for LNM in CRC, but also a functional protein that may play a significant role in cell migration. The proteins identified in the present study should further our understanding of the LNM process of CRC and may become useful markers for diagnosis and targets for therapeutic interventions. J. Cell. Biochem. 110: 1512–1519, 2010. © 2010 Wiley‐Liss, Inc.     

Chromatin accessibility changes are associated with enhanced growth and liver metastasis capacity of acid-adapted colorectal cancer cells

The acidic extracellular microenvironment, namely acidosis, is a biochemical hallmark of solid tumors. However, the tumorigenicity, metastatic potential, gene expression profile and chromatin accessibility of acidosis-adapted colorectal cancer cells remain unknown. The colorectal cancer cell SW620 was cultured in acidic medium (pH 6.5) for more than 3 months to be acidosis-adapted (SW620-AA). In comparison to parental cells, SW620-AA cells exhibit enhanced tumorigenicity and liver metastatic potential in vivo. Following mRNA and lncRNA expression profiling, we validated that OLMF1, NFIB, SMAD9, DGKB are upregulated, while SESN2, MAP1B, UTRN, PCDH19, IL18, LMO2, CNKSR3, GXYLT2 are downregulated in SW620-AA cells. The differentially expressed mRNAs were significantly enriched in DNA remodeling-associated pathways including HDACs deacetylate histones, SIRT1 pathway, DNA methylation, DNA bending complex, and RNA polymerase 1 chain elongation. Finally, chromatin accessibility evaluation by ATAC-sequencing revealed that the differentially opened peaks were enriched in pathways such as small cell lung cancer, pathways in cancer, ErbB signaling, endometrial cancer, and chronic myeloid leukemia, which were mainly distributed in intergenic regions and introns. These results suggest that the chromatin accessibility changes are correlated with enhanced growth and liver metastasis capacity of acid-adapted colorectal cancer cells.     

Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy

CD8⁺ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8⁺ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8⁺ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8⁺ T cells. Mechanically, Mn²⁺ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8⁺ T cell differentiation, activation and NK cell activation, and increased memory CD8⁺ T cells. Combining Mn²⁺ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn²⁺ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.Subject terms: Pattern recognition receptors, Immunosurveillance     

Metformin inhibition of colorectal cancer cell migration is associated with rebuilt adherens junctions and FAK downregulation

E-cadherin, a central component of the adherens junction (AJ), is a single-pass transmembrane protein that mediates cell–cell adhesion. The loss of E-cadherin surface expression, and therefore cell–cell adhesion, leads to increased cell migration and invasion. Treatment of colorectal cancer (CRC)-derived cells (SW-480 and HT-29) with 2.0 mM metformin promoted a redistribution of cytosolic E-cadherin to de novo formed puncta along the length of the contacting membranes of these cells. Metformin also promoted translocation from the cytosol to the plasma membrane of p120-catenin, another core component of the AJs. Furthermore, E-cadherin and p120-catenin colocalized with β-catenin at cell–cell contacts. Western blot analysis of lysates of CRC-derived cells revealed a substantial metformin-induced increase in the level of p120-catenin as well as E-cadherin phosphorylation on Ser^838/840, a modification associated with β-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and β-catenin localization suggest that metformin induces rebuilding of AJs in CRC-derived cells. Those modifications were accompanied by the inhibition of focal adhesion kinase (FAK), as revealed by a significant decrease in the phosphorylation of FAK at Tyr³⁹⁷ and paxillin at Tyr¹¹⁸. These changes were associated with a reduction in the numbers, but an increase in the size, of focal adhesions and by the inhibition of cell migration. Overall, these observations indicate that metformin targets multiple pathways associated with CRC development and progression.     

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

Objective Naturally occurring regulatory T (T_R) cells suppress autoreactive T cells whereas adaptive T_R cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T_R cells express COX-2 and produce PGE₂ that suppress effector T cells in a manner that is reversed by COX-inhibitors. Methods and results Here we demonstrate that CRC patients have elevated levels of PGE₂ in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T_R cells. Depletion of T_R cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity. Conclusion We suggest that adaptive T_R cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE₂-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T_R cells and the PGE₂–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.     

GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients

The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other “eat me” signals, it enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically-silent nature of apoptosis. Recently, however, PS exposure has also been reported in non-apoptotic forms of regulated inflammatory cell death, such as necroptosis, challenging previous dogma. In this review, we outline the evidence for PS exposure in non-apoptotic cells and extracellular vesicles (EVs), and discuss possible mechanisms based on our knowledge of apoptotic-PS exposure. In addition, we examine the outcomes of non-apoptotic PS exposure, including the reversibility of cell death, efferocytosis, and consequent inflammation. By examining PS biology, we challenge the established approach of distinguishing apoptosis from other cell death pathways by AnnexinV staining of PS externalization. Finally, we re-evaluate how PS exposure is thought to define apoptosis as an immunologically silent process distinct from other non-apoptotic and inflammatory cell death pathways. Ultimately, we suggest that a complete understanding of how regulated cell death processes affect the immune system is far from being fully elucidated.     

The impact on clinical outcome of high prevalence of diabetes mellitus in taiwanese patients with colorectal cancer

ABSTRACT: BACKGROUND: Both colorectal cancer (CRC) and diabetes mellitus (DM) are important public health problems worldwide. As there are controversies about survival impact on CRC patients with preexisting DM, the purpose of the present study is to evaluate the incidence and the survival impact of preexisting DM on the long-term outcomes of patients with CRC in Taiwan. METHODS: From January 2002 to December 2008, 1,197 consecutive patients with histologically proven primary CRC, who received surgical treatment at a single institution, were enrolled. The clinicopathologic features between these patients with and without DM were retrospectively investigated. Moreover, we intended to analyze the impact of DM on overall survival (OS) and cancer-specific survival (CSS) rates. RESULTS: Of 1,197 CRC patients, 23.6% of patients had either a reported history of DM or were currently taking one or more diabetes-controlling medications. CRC patients with DM were significantly older than those without DM (P <0.001), and had a higher incidence of cardiac disease and higher body mass index than those without DM (both P < 0.001). There were no significant differences in gender, tumor size, tumor location, histological type, AJCC/UICC cancer stage, vascular invasion, perineural invasion, comorbidity of pulmonary disease or renal disease, and OS, and CSS between two groups. Additionally, DM patients had a higher incidence of second malignancy than patients without DM (9.54% vs 6.01%, P = 0.040). CONCLUSIONS: A considerably high prevalence of DM in CRC patients but no significant impact of DM on survival was observed in the single-institution retrospective study, regardless of cancer stages and tumor locations. Therefore, treatment strategies for CRC patients with DM should be the same as patients without DM.     

Overexpression of insulin receptor substrate-4 is correlated with clinical staging in colorectal cancer patients

Insulin receptor substrate-4 (IRS-4), a poorly studied member of the IRS family, may play an important role in colorectal cancer (CRC) tumourigenesis. The aim of this pilot study was to elucidate the potential role of IRS-4 in colorectal carcinogenesis by evaluating IRS-4 expression in different types of colorectal tumours (n?=?20) and comparing its expression to normal mucosa (n?=?20). Tissue samples were collected from 18 patients with CRC and 2 with precancerous lesions (tubulovillous adenomas), all of whom were undergoing potentially curative surgery. IRS-4 expression was evaluated using immunohistochemical staining and compared to clinicopathological features. In normal colonic crypts, the subcellular localization of IRS-4 varied from the crypt base compartment to the surface epithelium. Nuclear IRS-4 staining decreased while non-nuclear IRS-4 increased as cells approached the top of the crypt. In the patients studied, colorectal tumours showed a significant (p < 0.001) increase of IRS-4 expression compared with adjacent normal tissue. Furthermore, nuclear IRS-4 intensity of CRC patients was significantly (p < 0.0001) higher in colonic tumoural tissue than in paired normal specimens. Tumour expression of IRS-4 in CRC patients was positively associated with T (p < 0.0001) and N (p < 0.05), of TNM (tumour and nodes and metastasis) staging system. Taken together, these results suggest that increase of IRS-4 expression may be involved to some extent in colorectal cancer.     

Identification of IMPA2 as the hub gene associated with colorectal cancer and liver metastasis by integrated bioinformatics analysis

Background and ObjectivesColorectal cancer (CRC) is one of the most common malignant tumors worldwide with high incidence and mortality rate, while colorectal liver metastasis (CRLM) is one of the major causes of cancer-related deaths. Therefore, the present study aims to identify the hub gene associated with CRC carcinogenesis and liver metastasis, and then explore its diagnostic and prognostic value as well as the potential regulation mechanism.MethodsThe overlapping differential co-expression genes among CRC, CRLM, and normal tissues were explored on the GSE49355 and GSE81582 datasets from the Gene Expression Omnibus (GEO) database by integrated bioinformatics analysis. Then, the hub prognostic genes were selected from the overlapping genes by univariate Cox proportional hazard analysis and online database Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Subsequently, the clinical value of the hub genes was evaluated in the TCGA and GSE39582 cohorts. Finally, the underlying mechanisms of the hub gene regulating CRC carcinogenesis and metastasis were explored by Gene function annotation and DNA methylation analysis.ResultsInositol mono-phosphatase 2 (IMPA2) was identified as the hub gene associated with CRC carcinogenesis and liver metastasis. IMPA2 had an excellent diagnostic efficiency, and its expression was significantly decreased in CRC and liver metastasis samples, being positively correlated with poor prognosis. Moreover, its low expression was associated with AJCC stage III+IV, T4, N1+2, and M1. In addition, our results revealed that the potential mechanisms used by IMPA2 to mediate CRC carcinogenesis and metastasis could be associated with lipid metabolism and epithelial mesenchymal transition (EMT). Finally, IMPA2 expression could be regulated by DNA methylation.ConclusionsIMPA2 was identified and reported for the first time as a hub gene biomarker in the diagnosis and prognosis of CRC, which could regulate CRC carcinogenesis and liver metastasis through the regulation of lipid metabolism, EMT, and DNA methylation.     

Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4)

We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.     

Decreased tumstatin-mRNA is associated with poor outcome in patients with NSCLC

Tumstatin is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. The purpose of this study was to evaluate the correlation between tumstatin-mRNA expression and the clinicopathologic characteristics, tumor angiogenesis, outcome of patients with non-small cell lung cancer (NSCLC). Specimens from 68 patients with NSCLC were recruited in this study. Tumstatin-mRNA expression and protein level in tumor tissues were quantified respectively by quantitative RT-PCR and ELISA. Microvessel density (MVD) was determined by CD34 immunostaining. The correlation of tumstatin-mRNA expression levels with clinicopathologic variables, tumor angiogenesis, and prognosis was analyzed. Tumstatin-mRNA expression levels were decreased in tumor. Tumstatin-mRNA expression level was significantly correlated with its protein level in tumor (r = 0.562; P = 0.001). Tumstatin-mRNA expression levels in poorly differentiated tumor tissues were significantly lower than in well-differentiated tumor tissues (P < 0.004). Furthermore, tumor tumstatin-mRNA expression were also significantly related to tumor pathologic stage (P = 0.032) and MVD (r = -0.77, P < 0.0001). Overall survival (OS) and disease-free survival (DFS) analysis indicated that NSCLC patients with low tumstatin-mRNA expression had poorer OS and DFS than those with high expression (P = 0.015 and 0.037; respectively). Multivariable Cox regression analysis revealed that the tumstatin-mRNA expression could be an independent prognostic indicators in both DFS and OS. Tumstatin-mRNA expression levels are down-regulated in NSCLC tissues. Tumstatin-mRNA expression level correlates with prognosis, which suggests that tumstatin-mRNA is a new potential independent marker of favorable prognosis in NSCLC.     

Overexpression of LI-cadherin in gastric cancer is associated with lymph node metastasis

Gastric cancer remains the second leading cause of cancer deaths worldwide. Patients usually present late with local invasion or metastatic diseases. The present study investigated the expression level of liver-intestine cadherin (LI-cadherin) by RT-PCR and its correlation with clinicopathological data in 71 pairs of tumor and non-cancerous gastric mucosa. Protein expression level of LI-cadherin was determined by Western blotting and immunohistochemistry. The mRNA of LI-cadherin was highly expressed in tumor as compared to non-cancerous mucosa. Lymph node metastasis was significantly associated with the expression of LI-cadherin (p=0.038). On multivariate analysis, T staging and LI-cadherin expression were found to be independent factors associated with lymph node metastasis.     

SLAMF8, a potential new immune checkpoint molecule,is associated with the prognosis of colorectal cancer

Recently, immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1) monoclonal antibodies (mAbs), have revolutionized the treatment of malignant tumors. Therefore, the number of studies aiming to screen and identify new immune checkpoint molecules for antitumor immunotherapy is increasing. Signaling lymphocytic activation molecule (SLAM) family members are mainly expressed by and regulate the functions of immune cells. Recent studies have shown that several SLAM family members are involved in the regulation of the tumor immune microenvironment and are promising targets for antitumor immunotherapy. Signaling lymphocytic activation molecule family member 8 (SLAMF8) is a type I cell surface glycoprotein and is encoded on chromosome 1q21. To further illustrate the clinical value of SLAMF8 in colorectal cancer (CRC), we retrospectively analyzed the relationship between SLAMF8 expression and the prognosis of CRC patients and the associations between SLAMF8 expression and the expression levels of other SLAM family members and other classic immune checkpoint molecules using The Cancer Genome Atlas (TCGA) data, RNA sequencing data, tissue immunohistochemistry staining, and systematic follow-up analysis. Here, high SLAMF8 expression was associated with poor overall survival (OS) in CRC. The mRNA expression level of SLAMF8 was positively correlated with the expression levels of multiple classic immune checkpoints and other SLAM family members. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the pathways enriched in CRC tissues with high SLAMF8 expression were associated with the regulation of the tumor immune microenvironment.